Protein having B-glucosidase activity and uses thereof

ABSTRACT

By combination of hydrophobic chromatography and strongly basic anion-exchange chromatography, a novel, highly hydrophobic β-glucosidase was successfully identified from Acremonium cellulolyticus. Further, a gene corresponding to the identified β-glucosidase was isolated. When multiple modifications were introduced into the base sequence of the gene, the gene was successfully expressed in Trichoderma viride at a high level, and the expression product successfully exhibited a high β-glucosidase activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.13/391,598, filed Feb. 21, 2012 (now allowed); which is a National Stageof International Application No. PCT/JP2010/063844 filed Aug. 17, 2010;which claims priority based on Japanese Patent Application No.2009-190840 filed Aug. 20, 2009; the contents of all of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a novel protein having a β-glucosidaseactivity and uses thereof. Specifically, the present invention relatesto a novel protein having a β-glucosidase activity derived fromAcremonium cellulolyticus, an analog and a variant of the protein,polynucleotides encoding these proteins, and a production method for anduses of these proteins.

BACKGROUND ART

Cellulose is an essential constitutive component of cells of higherplants, and widely exists in nature. Cellulose is a polysaccharidepolymer of glucose units polymerized through a β-1,4-glycosidic bond. Innature, cellulose exists in a crystalline or amorphous state. By bondingto other components such as lignin, hemicelluloses, and pectins in acomplicated manner, cellulose constructs plant tissues.

Cellulase is a generic term of a group of enzymes that breaks downcellulose. Generally, cellulase produced by microorganisms includesvarious types of cellulase components. In accordance with the substratespecificity, the cellulase components are classified into three types:cellobiohydrolase, endoglucanase, and β-glucosidase. Aspergillus nigerthat is a cellulase-producing filamentous fungus is believed to produce4 types of cellobiohydrolases at maximum, 15 types of endoglucanases,and 15 types of β-glucosidases. Presumably, multiple enzymes among theseacting in various reaction modes compensate for each other to exhibitsynergistic effects, thereby breaking down cellulose which is anessential component of plant cell walls. It is believed thatβ-glucosidase catalyzes a reaction to release glucose fromcello-oligosaccharides, cellobiose or glycosides with aglycone linkedthereto through β-D-glucopyranosyl linkage. β-glucosidase is animportant enzyme in the final stage of cellulose saccharification and inreleasing glucose from glycoside.

Ethanol conversion from biomass has advantages that: the raw material ismore readily available, combustion of the raw material or burying in theground can be avoided, and ethanol fuel is environmentally clean. Woods,agricultural residues, herbaceous crops and municipal solid wastes havedrawn attention as biomass for ethanol production. These materials aremainly composed of cellulose, hemicellulose and lignin. Once celluloseis converted into glucose, the glucose is easily fermented into ethanolby yeasts. On the other hand, cellobiose is not easily fermented intoethanol by yeasts, and accordingly the remaining cellobiose causesethanol yield reduction. What is more important is that cellobiose is apotent inhibitor of endoglucanases and cellobiohydrolases. For thisreason, the accumulation of cellobiose during hydrolysis is notdesirable for production of ethanol. Generally, cellulase-producingmicroorganisms can hardly produce β-glucosidases. This brings about amajor problem that cellobiose produced by enzymatic hydrolysis isaccumulated.

In order to promote conversion from cellobiose to glucoses, the yield ofβ-glucosidases is increased by over-expression of β-glucosidases in ahost. Thus, it is effective means for promoting saccharification frombiomass to glucose. Accordingly, isolation of a novel β-glucosidase genewhich is introduced and expressed in cellulase-producing microorganismshas been desired.

Meanwhile, filamentous fungus Acremonium cellulolyticus has beenreported to produce a cellulase having a strong saccharification power(Non Patent Literature 1) and to be highly useful for feed and silageusages (Patent Literatures 1 to 3). In addition, the cellulase componentcontained therein (Patent Literatures 4 to 10) has also been examined indetail. It has been revealed that various kinds of cellulase componentare secreted as in other filamentous fungi. Particularly, as to theβ-glucosidase activity of the cellulase therein, it has been reported,for example, that the activity is significantly higher than those ofcellulases from Trichoderma reesei and the like (Patent Literature 11).Because of such characteristics, attention has been focused onAcremonium cellulolyticus as the target from which a β-glucosidase geneis isolated.

However, only a few genes have been isolated from Acremoniumcellulolyticus so far (Patent Literatures 9, 10). Further, the isolatedgenes have not yet been successfully expressed in filamentous fungiother than Acremonium cellulolyticus.

CITATION LIST Patent Literatures

-   -   [PTL 1] Japanese Unexamined Patent Application Publication No.        Hei 7-264994    -   [PTL 2] Japanese Patent No. 2531595    -   [PTL 3] Japanese Unexamined Patent Application Publication No.        Hei 7-236431    -   [PTL 4] Japanese Unexamined Patent Application Publication No.        2001-17180    -   [PTL 5] International Publication No. WO97/33982    -   [PTL 6] International Publication No. WO99/011767    -   [PTL 7] Japanese Unexamined Patent Application Publication No.        2000/69978    -   [PTL 8] Japanese Unexamined Patent Application Publication No.        Hei 10-066569    -   [PTL 9] Japanese Unexamined Patent Application Publication No.        2002/101876    -   [PTL 10] International Publication No. WO2002/026979    -   [PTL 11] Japanese Examined Patent Application Publication No.        Sho 60-43954

Non Patent Literature

-   -   [NPL 1] “Agricultural and Biological Chemistry,” (Japan), 1987,        Vol. 51, p. 65

SUMMARY OF INVENTION Technical Problem

The present invention has been made in view of such circumstances. Anobject of the present invention is to isolate a novel β-glucosidase genefrom Acremonium cellulolyticus. Another object of the present inventionis to increase the yield of β-glucosidase from a host in which theisolated β-glucosidase gene is expressed at a high level.

Solution to Problem

To achieve the above-described objects, the present inventors haveearnestly studied methods of separating and purifying a β-glucosidasederived from Acremonium cellulolyticus. As a result, the inventors havefinally successfully identified a novel β-glucosidase different fromβ-glucosidases that have been known so far from Acremoniumcellulolyticus. Moreover, a gene encoding the identified β-glucosidasehas also been successfully isolated. The β-glucosidase gene found out bythe present inventors had not been isolated despite attempts over theyears to isolate the gene from Acremonium cellulolyticus. The reason ispresumably that since the hydrophobicity of a protein encoded by thegene is high, this makes the separation and purification thereofdifficult.

Further, the present inventors have earnestly studied methods ofexpressing the isolated β-glucosidase gene derived from Acremoniumcellulolyticus at a high level in a host, thereby causing the host toproduce a β-glucosidase having an excellent activity. As a result, byintroducing modification of multiple bases into the β-glucosidase gene,successfully, for the first time in the world, high-level expression ofa β-glucosidase gene was achieved in filamentous fungi other thanAcremonium cellulolyticus, and the expression product exhibited a highβ-glucosidase activity. This makes it possible to express aβ-glucosidase derived from Acremonium cellulolyticus at a high level ina host, and to increase an amount of β-glucosidase produced. The presentinventors have found out that by using the β-glucosidase or a cellulasepreparation obtained from the transformant thus prepared,saccharification from biomass to glucose and various treatments andmodifications on a cellulose-based substrate can be efficiently carriedout. This discovery has led to the completion of the present invention.

Specifically, the present invention relates to a novel protein having aβ-glucosidase activity derived from Acremonium cellulolyticus, an analogand a variant of the protein, polynucleotides encoding these proteins,and a production method for and uses of these proteins. Morespecifically, the present invention provides the followings.

-   (1) A polynucleotide of any one of the following (i) to (vi), which    encodes a protein having a β-glucosidase activity:

(i) a polynucleotide encoding a protein comprising an amino acidsequence of SEQ ID NO: 3;

(ii) a polynucleotide comprising a coding region of a base sequence ofany one of SEQ ID NOs: 1 and 2;

(iii) a polynucleotide encoding a protein comprising an amino acidsequence of SEQ ID NO: 3 in which one or more amino acids aresubstituted, deleted, inserted and/or added;

(iv) a polynucleotide encoding a protein comprising an amino acidsequence having an identity of 90% or more with the amino acid sequenceof SEQ ID NO: 3;

(v) a polynucleotide hybridizing under stringent conditions with apolynucleotide comprising the base sequence of any one of SEQ ID NOs: 1and 2; and

(vi) a polynucleotide of any one of (i) to (v) from which a basesequence encoding a signal sequence is removed.

-   (2) The polynucleotide according to (1), which is derived from a    filamentous fungus.-   (3) The polynucleotide according to (2), wherein the filamentous    fungus is Acremonium cellulolyticus.-   (4) A polynucleotide of any one of the following (i) and (ii), which    encodes a protein having a β-glucosidase activity:

(i) a polynucleotide encoding a protein comprising an amino acidsequence of SEQ ID NO: 3; and

(ii) a polynucleotide comprising a coding region of a base sequence ofSEQ ID NO: 4.

-   (5) A polynucleotide comprising a base sequence of SEQ ID NO: 4 in    which one or more bases are substituted, deleted, inserted and/or    added, the polynucleotide encoding a protein having a β-glucosidase    activity and being expressible in Trichoderma viride.-   (6) The polynucleotide according to (5), being capable of improving    a β-glucosidase activity when expressed in a Trichoderma viride    transformant 5 times or more in comparison with a β-glucosidase    activity in Trichoderma viride parental strain.-   (7) The polynucleotide according to any one of (4) to (6), from    which a base sequence encoding a signal sequence is removed.-   (8) An expression vector comprising the polynucleotide according to    any one of (1) to (7).-   (9) A host cell transformed with the expression vector according to    (8).-   (10) A protein encoded by the polynucleotide according to any one    of (1) to (7).-   (11) The protein according to (10), which is a recombinant protein.-   (12) A method for producing the protein according to (11), the    method comprising the steps of:

culturing the host cell according to (9); and

harvesting a protein expressed from the host cell and/or a culturethereof.

-   (13) A cellulase preparation comprising the protein according to    (11).-   (14) A method for degrading or converting a cellulose material, the    method comprising the steps of:

treating the cellulose material with any one of the protein according to(10) and the cellulase preparation according to (13).

-   (15) A method for producing a degraded or converted cellulose    material, the method comprising the steps of:

treating a cellulose material with any one of the protein according to(10) and the cellulase preparation according to (13); and

collecting a degraded cellulose material.

-   (16) The method according to (15), wherein the degraded cellulose    material is a sugar.-   (17) A detergent composition comprising any one of the protein    according to (10) and the cellulase preparation according to (13).-   (18) A method for treating a cellulose-containing fiber, the method    comprising the steps of:

bringing into contact any one of the protein according to (10), thecellulase preparation according to (13), and detergent composition theaccording to (17) with the cellulose-containing fiber.

-   (19) A method for deinking waste paper, the method characterized in    that any one of the protein according to (10) and the cellulase    preparation according to (13) is used in a deinking step of treating    the waste paper with a deinking agent.-   (20) A method for producing paper pulp having an improved drainage,    the method comprising the steps of:

treating paper pulp with any one of the protein according to (10) andthe cellulase preparation according to (13).

-   (21) A method for producing an animal feed having an improved    digestibility, the method comprising the step of:

treating an animal feed with any one of the protein according to (10)and the cellulase preparation according to (13).

-   (22) A filamentous fungus expressing a reduced amount of a protein    encoded by the polynucleotide according to (2).

Advantageous Effects of Invention

The present invention provides a novel β-glucosidase gene derived fromAcremonium cellulolyticus, and an analog and a variant of the gene forefficiently expressing a β-glucosidase in a host. Further, the presentinvention provides a host which expresses the β-glucosidase at a highlevel, and which shows an excellent β-glucosidase activity. This makesit possible to obtain a β-glucosidase derived from Acremoniumcellulolyticus as a purified protein or a cellulase preparation in highyield.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 is a scheme showing a restriction enzyme map of a plasmid pBGLB.

DESCRIPTION OF EMBODIMENTS

Protein Having β-Glucosidase Activity and Polynucleotide Encoding theProtein

The present invention provides a novel protein having a β-glucosidaseactivity and a polynucleotide encoding the protein. In the presentinvention, the “β-glucosidase” means an enzyme showing a β-glucosidaseactivity, that is, β-D-Glucoside glucohydrolase EC3.2.1.21. The“β-glucosidase activity” means an activity of hydrolyzingcello-oligosaccharides, cellobiose, or glycosides with aglycone linkedthereto through β-D-glucopyranosyl linkage by an exo-mechanism toproduce glucose.

The “polynucleotide” encoding the protein having a β-glucosidaseactivity of the present invention includes, for example, a DNA, a RNA,modified products or chimeras thereof, and is preferably a DNA. The DNAincludes a cDNA, a genomic DNA, and a chemically synthesized DNA. Thebase sequence of a cDNA which is isolated by the present inventors, andwhich encodes the novel β-glucosidase (hereinafter, referred to as“acBGLB”) derived from Acremonium cellulolyticus, is shown in SEQ IDNO: 1. The base sequence of the genomic DNA is shown in SEQ ID NO: 2.Moreover, the amino acid sequence of the acBGLB encoded by these DNAs isshown in SEQ ID NO: 3.

A preferable embodiment of the polynucleotide of the present inventionis a polynucleotide encoding the acBGLB comprising the amino acidsequence of SEQ ID NO: 3. An example thereof includes a polynucleotidecomprising a coding region of the base sequence of any one of SEQ IDNOs: 1 and 2.

Moreover, the present invention comprises a polynucleotide encoding aprotein functionally equivalent to the acBGLB. Examples of such apolynucleotide include mutants, derivatives, alleles, variants andhomologs of the acBGLB. Herein, the phrase “functionally equivalent”means that the target protein has a β-glucosidase activity. Preferably,when compared, the target protein has 70% or more, preferably 80% ormore, more preferably 90% or more, and most preferably 95% or more ofthe β-glucosidase activity of the acBGLB. The β-glucosidase activity ofthe target protein and the acBGLB can be evaluated as an activity ofproducing 1 μmol of p-nitrophenol from p-nitrophenyl-β-glucoside in 1minute when measured by a method described in the literature (Methods inENZYMOLOGY, vol. 160, Biomass Part A Cellulose and Hemicellulose, WillisA. Wood ed. p 109-110).

One embodiment of the polynucleotide encoding a protein functionallyequivalent to the acBGLB is a polynucleotide encoding a protein having aβ-glucosidase activity, the protein comprising an amino acid sequence ofSEQ ID NO: 3 in which one or more amino acids are substituted, deleted,and/or added.

The number of amino acid residues modified is preferably 1 to 40, morepreferably 1 to 20, further preferably 1 to 8, and most preferably 1 to4. The modification of amino acids is preferably conservativesubstitution. The “conservative substitution” means that at least oneamino acid residue is substituted with another chemically similar aminoacid residue in such a manner as not to substantially change theactivity of the polypeptide. Examples thereof include a case where acertain hydrophobic amino acid residue is substituted with anotherhydrophobic amino acid residue, a case where a certain polar amino acidresidue is substituted with another polar amino acid residue having thesame charge, and other cases. Functionally similar amino acids which canbe subjected to such substitution are known to those skilled in the artfor each amino acid. Specific examples of non-polar (hydrophobic) aminoacids include alanine, valine, isoleucine, leucine, proline, tryptophan,phenylalanine, methionine, and the like. Specific examples of polar(neutral) amino acids include glycine, serine, threonine, tyrosine,glutamine, asparagine, cysteine, and the like. Specific examples ofpositively-charged (basic) amino acids include arginine, histidine,lysine, and the like. Further, specific examples of negatively-charged(acidic) amino acids include aspartic acid, glutamic acid, and the like.

The polynucleotide encoding the protein having a β-glucosidase activityof the present invention is preferably a polynucleotide encoding aprotein comprising an amino acid sequence of SEQ ID NO: 3 (for example,a polynucleotide comprising a coding region of a base sequence of SEQ IDNO: 4), particularly when expressed in Trichoderma viride. In the basesequence of SEQ ID NO: 4, 13.2% or more of the bases are modified incomparison with the base sequence (SEQ ID NO: 1) of the polynucleotideencoding the acBGLB. In determining a codon corresponding to each aminoacid, the frequency distribution of codons used in a host is taken intoconsideration. This enables the expression in Trichoderma viride, andthe expression product successfully exhibits a high β-glucosidaseactivity. Once such a preferable sequence is obtained, those skilled inthe art could, on the basis of this sequence, further modify the basesequence to obtain a polynucleotide expressible in Trichoderma in thesame manner as the polynucleotide comprising the coding region of thebase sequence of SEQ ID NO: 4. Thus, the present invention provides apolynucleotide comprising the base sequence of SEQ ID NO: 4 in which oneor more bases (preferably 30 bases or less, more preferably 20 bases orless, further preferably 10 bases or less, and still further preferably5 bases or less) are substituted, deleted, inserted and/or added, thepolynucleotide encoding a protein having a β-glucosidase activity andbeing expressible in Trichoderma viride. A preferable embodiment of sucha polynucleotide is a polynucleotide capable of improving aβ-glucosidase activity when expressed in a Trichoderma viridetransformant 5 times or more (preferably, 7 times or more) in comparisonwith a β-glucosidase activity in a Trichoderma viride parental strain(original Trichoderma viride strain not deficient in a gene for uracilbiosynthesis) (see Example 5).

In the present invention, another embodiment of the polynucleotideencoding a protein functionally equivalent to the acBGLB is apolynucleotide encoding a protein having a β-glucosidase activity, theprotein comprising an amino acid sequence having an identity of 90% ormore with the amino acid sequence of SEQ ID NO: 3. Herein, the“identity” is a numerical value calculated by using the default (initialsetting) parameters in FASTA3 [Science, 227, 1435-1441(1985), Proc.Natl. Acad. Sci. USA, 85, 2444-2448 (1988),http://www.ddbj.nig.ac.jp/E-mail/homology-j.html] that is a homologysearch program known to those skilled in the art. The identity can bepreferably an identity of 95% or more, further preferably an identity of98% or more, and particularly preferably an identity of 99% or more.

In the present invention, another embodiment of the polynucleotideencoding a protein functionally equivalent to the acBGLB is apolynucleotide encoding a protein having a β-glucosidase activity, thepolynucleotide hybridizing under stringent conditions with apolynucleotide comprising the base sequence of any one of SEQ ID NOs: 1and 2. Herein, the “stringent conditions” mean that under which amembrane washing procedure after the hybridization is carried out athigh temperature in a solution having a low salt concentration, andmeans washing conditions, for example, at 2×SSC concentration (1×SSC: 15mmol/L of trisodium citrate and 150 mmol/L of sodium chloride) and in a0.5% SDS solution at 60° C. for 20 minutes.

Further, the present invention provides a polynucleotide encoding theacBGLB or a protein functionally equivalent thereto, from which a basesequence encoding a signal sequence is removed. The signal sequence ofthe acBGLB is an amino acid sequence from positions −18 to −1 in theamino acid sequence of SEQ ID NO: 3.

Any polypeptide sequence may be added to the N-terminus and/or theC-terminus of each amino acid sequence corresponding to a mature proteinportion of the protein of the present invention in such a manner as notto influence the β-glucosidase activity. Examples of such a polypeptidesequence include a signal sequence, a detection marker (for example, aFLAG tag), and a purification polypeptide [for example, glutathioneS-transferase (GST)].

The polynucleotide encoding the protein having a β-glucosidase activityof the present invention can be prepared by utilizing conventional meansfor those skilled in the art. In preparing a genomic DNA encoding theprotein having a β-glucosidase activity of the present invention, forexample, first, a genomic DNA is extracted from a target microorganismsuch as Acremonium cellulolyticus by a generally-used method. Thegenomic DNA is digested with an appropriate restriction enzyme and thenligated to an appropriate vector. Thereby, a genomic DNA library ofAcremonium cellulolyticus is prepared. As the vector, various vectorscan be used such as, for example, a plasmid vector, a phage vector, acosmid vector, and a BAC vector. Next, an appropriate probe is preparedbased on the base sequence (for example, SEQ ID NO: 2) of thepolynucleotide encoding the protein having a β-glucosidase activity ofthe present invention, and a desired genomic DNA can be isolated fromthe genomic DNA library through hybridization. Alternatively, a primeris prepared based on the base sequence (for example, SEQ ID NO: 2) ofthe polynucleotide encoding the protein having a β-glucosidase activityof the present invention, and PCR is performed using the genomic DNA ofAcremonium cellulolyticus as a template. A DNA fragment thus amplifiedis ligated to an appropriate vector. Accordingly, a desired genomic DNAcan be isolated. Meanwhile, in preparing a cDNA encoding the proteinhaving a β-glucosidase activity of the present invention, for example,first, a cDNA is synthesized based on an mRNA extracted from a targetmicroorganism such as Acremonium cellulolyticus. The cDNA is digestedwith an appropriate restriction enzyme and then ligated to anappropriate vector. Thereby, a cDNA library of Acremonium cellulolyticusis prepared. Next, an appropriate probe is prepared based on the basesequence (for example, SEQ ID NO: 1) of the polynucleotide encoding theprotein having a β-glucosidase activity of the present invention, and adesired cDNA can be isolated from the cDNA library throughhybridization. Alternatively, a primer is prepared based on the basesequence (for example, SEQ ID NO: 1) of the polynucleotide encoding theprotein having a β-glucosidase activity of the present invention, andPCR is performed using the cDNA of Acremonium cellulolyticus as atemplate. A DNA fragment thus amplified is ligated to an appropriatevector. Accordingly, a desired cDNA can be isolated. Furthermore, thepolynucleotide encoding the protein having a β-glucosidase activity ofthe present invention can be obtained artificially by chemicalsynthesis.

The present invention provides an expression vector comprising: thepolynucleotide encoding the protein having a β-glucosidase activity ofthe present invention, the polynucleotide being replicable in a hostmicroorganism; and the protein encoded from the polynucleotide sequencein an expressible state. The expression vector of the present inventioncan be constructed based on a self-replicating vector, i.e., forexample, a plasmid which exists as an extrachromosomal element, andwhich replicates independently of the replication of the chromosome.Alternatively, the expression vector of the present invention may bereplicated together with the chromosome of the host microorganism, afterintroduced into the host microorganism and incorporated into the genomethereof. As a procedure and a method for constructing the expressionvector of the present invention, any procedure and any method commonlyused in the field of genetic engineering can be used.

To express the protein having a β-glucosidase activity after theexpression vector according to the present invention is actuallyintroduced in a host microorganism, the expression vector according tothe present invention desirably comprises, in addition to thepolynucleotide encoding the protein having a β-glucosidase activity ofthe present invention, a polynucleotide sequence for regulating theexpression, a genetic marker for selecting a microorganism, and thelike. Examples of the polynucleotide sequence for regulating theexpression include polynucleotide sequences encoding a promoter,terminator, and signal peptide. The promoter is not particularlylimited, as long as the transcriptional activity is exhibited in thehost microorganism. The promoter may be derived from a microorganismeither homologous or heterologous to the host microorganism. Moreover,the signal peptide is not particularly limited, as long as the signalpeptide contributes to secretion of the protein in the hostmicroorganism. The signal peptide may be derived from a microorganismeither homologous or heterologous to the host microorganism. Further,the genetic marker can be selected as appropriate according to themethod of selecting the transformant. For example, a gene encoding drugresistance or a gene complementing the auxotrophy can be used.

Further, the present invention provides a microorganism trans formedwith the expression vector. The host microorganism used in the presentinvention is not particularly limited. Examples thereof includefilamentous fungi, yeasts, Escherichia coli, actinomycetes, and thelike. Examples of the yeast cells include those belonging to the generaSaccharomyces, Hansenula, and Pichia. A preferable example of the yeastcell is Saccharomyces cerevisiae. Moreover, examples of the filamentousfungi include those belonging to the genera Humicola, Aspergillus,Trichoderma, Fusarium, and Acremonium. Preferably examples of thefilamentous fungi include Humicola insolens, Aspergillus niger,Aspergillus oryzae, Trichoderma viride, Fusarium oxysporum, andAcremonium cellulolyticus. The transformation of these microorganismswith the expression vector of the present invention can be carried outaccording to any method commonly used in this field.

The protein having a β-glucosidase activity of the present invention (ora cellulase preparation of the present invention to be described later)can be collected from a culture (for example, cultured cell, culturesupernatant) obtained by culturing the thus-prepared transformant in anappropriate medium. The culturing of the transformant and conditionstherefor may be substantially the same as those for a microorganism tobe used. Moreover, as the method of collecting the target protein afterthe transformant is cultured, any method commonly used in this field canbe used. For example, after the culturing of the transformant isfinished, a supernatant fluid obtained by removal from the culture bycentrifugation or the like can be used as a crude enzyme. Further, thissupernatant fluid is concentrated by an ultrafiltration method or thelike, and an antiseptic and the like are added thereto. The resultantcan be used as a concentrated enzyme. Furthermore, after theconcentrating, a powdery enzyme can be prepared by a spray-dry method orthe like. The protein having a β-glucosidase activity of the presentinvention (or the cellulase preparation of the present invention) can beobtained, as necessary, by partially purifying or highly purifying theconcentrated enzyme or the powdery enzyme. As the purification method,conventional methods, for example, a salting-out method with ammoniumsulfate or the like, an organic solvent precipitation method withalcohol or the like, a membrane separation method, and a chromatographicseparation method using an ion exchanger, a hydrophobic chromatographycarrier, a gel filtration carrier, or the like, can be used alone or incombination as appropriate. The present invention provides such a methodfor producing the protein having a β-glucosidase activity of the presentinvention (or the cellulase preparation of the present invention).

Cellulase Preparation

The present invention provides a cellulase preparation comprising theabove-described protein having a β-glucosidase activity of the presentinvention. The cellulase preparation of the present invention maycomprise a different protein from the protein having a β-glucosidaseactivity of the present invention. As the different protein, thecellulase preparation of the present invention may comprise, forexample, a β-glucosidase other than the protein having a β-glucosidaseactivity of the present invention, hemicellulase, endoglucanase,cellobiohydrolase, aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, α-galactosidase,β-galactosidase, glucoamylase, α-glucosidase, haloperoxidase, invertase,laccase, lipase, mannosidase, oxidase, pectinase, peptide glutaminase,peroxidase, phytase, polyphenol oxidase, protease, ribonuclease,transglutaminase, orxylanase. The protein different from the proteinhaving a β-glucosidase activity of the present invention comprised inthe cellulase preparation of the present invention may be derived fromthe transformant which expresses the protein having a β-glucosidaseactivity of the present invention or may be one independently added.

The cellulase preparation of the present invention may be produced whilebeing mixed with generally-contained carrier or medium, for example, anexcipient (for example, lactose, sodium chloride, sorbitol, or thelike), a surfactant, an antiseptic, or the like. Moreover, the cellulasepreparation of the present invention can be produced in an appropriateform, for example, powder or liquid.

Uses of Protein Having β-Glucosidase Activity or Cellulase Preparation

The present invention provides a method for degrading or converting acellulose material, the method comprising: treating the cellulosematerial with any one of the protein having a β-glucosidase activity ofthe present invention and the cellulase preparation of the presentinvention. Moreover, the present invention provides a method forproducing a degraded or converted cellulose material, the methodcomprising: treating a cellulose material; and then collecting adegraded cellulose material. The cellulose material is typically abiomass. Examples thereof include, but are not limited to, rice straw,bagasse, corn stover, pomaces of fruits such as coconut, waste wood, andmaterials obtained by subjecting these to an appropriate pre-treatment.The protein having a β-glucosidase activity or the cellulase preparationused for treating the cellulose material may be in the form of a crudefermentation broth from which cells are removed or not removed, or maybe in the form of semi-purified or purified preparation. In thefermentation process using the biomass, the transformant of the presentinvention can be used as a source of producing the protein having aβ-glucosidase activity of the present invention. To the transformant,various cellulase genes or a gene encoding another enzyme effective inprocessing the biomass may be introduced. The methods of the presentinvention can be utilized to produce a sugar (for example,monosaccharides, disaccharides, polysaccharides) as chemical orfermentation feedstocks from the biomass, for example. The sugar thusobtained serves as a raw material for producing, for example, ethanol,plastics, other products or intermediates.

Further, the present invention provides a detergent compositioncomprising any one of the protein having a β-glucosidase activity of thepresent invention and the cellulase preparation of the presentinvention. The detergent composition of the present invention may alsocomprise a surfactant (anionic, nonionic, cationic, amphoteric orzwitterionic surfactant, or may be a mixture of these). Moreover, thedetergent composition may also comprise other detergent components knownin this field, for example, a builder, a bleach, a bleach activator, acorrosion inhibitor, a sequestering agent, a soil release polymer, aflavor, other enzymes (protease, lipase, amylase, and the like), anenzyme stabilizer, a formulation aid, an optical brighter, and/or afoaming agent, and the like.

Furthermore, the present invention provides a method for treating acellulose-containing fiber, the method comprising the steps of bringingany one of the protein having a β-glucosidase activity of the presentinvention, the cellulase preparation of the present invention, and thedetergent composition into contact with the cellulose-containing fiber.Examples of characteristics of the cellulose-containing fiber to beimproved by the treatment method of the present invention include: (1)touch feel and appearance of the fiber improved by the reduced weight,(2) local variations in color provided to the coloredcellulose-containing fiber, that is, stonewashed appearance and textureprovided to the colored cellulose-containing fiber, typically jeans, (3)clearness of the color of the colored cellulose-containing fiber, (4)softness (slowed timing when the material starts to stiffen, reductionin stiffness), and (5) removal of fuzz (slowed timing when fuzz startsto form, reduction in fuzz).

Furthermore, the present invention provides a method for deinking wastepaper, the method characterized in that any one of the protein having aβ-glucosidase activity of the present invention and the cellulasepreparation of the present invention is used in a deinking step oftreating the waste paper with a deinking agent.

Furthermore, the present invention provides a method for producing paperpulp having an improved drainage, the method comprising the step of:treating paper pulp with any one of the protein having a β-glucosidaseactivity of the present invention and the cellulase preparation of thepresent invention. According to the present invention, the drainage ofthe paper pulp can be improved without a significant reduction instrength. Examples of the paper pulp that is the treatment targetinclude, but are not limited to, waste paper pulp, recycled paperboardpulp, Kraft pulp, sulfite pulp, thermo-mechanically treated and otherhigh yield pulps.

Furthermore, the present invention provides a method for producing ananimal feed having an improved digestibility, the method comprising thestep of: treating an animal feed with any one of the protein having aβ-glucosidase activity of the present invention and the cellulasepreparation of the present invention. According to the method of thepresent invention, the digestibility of glucan in the feed in an animalbody can be improved, for example.

Filamentous Fungus Expressing Reduced Amount of Protein Havingβ-Glucosidase Activity

The present invention provides a filamentous fungus expressing a reducedamount of the protein having a β-glucosidase activity of the presentinvention. The filamentous fungus is preferably a filamentous fungusbelonging to the genus Acremonium, and most preferably Acremoniumcellulolyticus. The expression of the protein having a β-glucosidaseactivity of the present invention (endogenous protein) in thefilamentous fungus can be reduced by utilizing general techniques suchas, for example, RNA interference method, antisense RNA⋅DNA method, andhomologous recombination. Methods of preparing polynucleotide molecules(for example, a siRNA, an antisense RNA, an antisense DNA, apolynucleotide comprising a sequence homologous to a target DNA for therecombination, and the like) used in these techniques, preparing vectorscomprising these polynucleotides, and introducing the vectors into hostsare known to those skilled in the art. When cellulose widely distributedamong plants and so forth is degraded by using the filamentous fungusthus produced, glucose which is a final degradation product in thedegradation process is not produced, but cellobiose in which two glucosemolecules are linked by a β-1,4 bond is selectively produced. Cellobiosehas a sweet taste but is not degraded in human bodies. Accordingly,cellobiose is useful as a sweetener in health food and food for diabeticpatients, a cosmetic raw material, or a drug raw material. By utilizingthe filamentous fungus of the present invention, the raw materials ofthese products can be provided at reduced cost.

EXAMPLES

The present invention will be more specifically described by way ofExamples. However, the present invention is not to be limited toExamples below but is still within the gist of the present invention.

Example 1 Purification of β-Glucosidase of Acremonium cellulolyticus

A spray-dried powdery enzyme of cellulase was prepared from Acremoniumcellulolyticus, and dissolved in a Tris-HCl buffer (0.05 M, pH 7.0)containing 0.5 M (NH₄)₂SO₄. Impurities were removed therefrom byhigh-performance cooling centrifugation. A supernatant thus obtained waspurified as a starting material for the enzyme purification according toa method shown below.

(a) Hydrophobic Chromatography (Part 1)

In a Tris-HCl buffer (0.05 M, pH 7.0) containing 0.5 M (NH₄)₂SO₄, aprotein contained in the supernatant was adsorbed to HiTrap Butyl FF(manufactured by GE Healthcare). Then, in a Tris-HCl buffer (0.05 M, pH7.0) containing 0.5 M to 0 M of (NH₄)₂SO₄, the adsorbed protein wassubjected to linear gradient elution to fractionate a fractionindicating a β-glucosidase activity.

(b) Hydrophobic Chromatography (Part 2)

A protein in the fraction obtained in (a) above was again adsorbed toHiTrap Butyl FF (manufactured by GE Healthcare). Then, the adsorbedprotein was subjected to linear gradient elution by the same method asin (a) to fractionate a fraction indicating a β-glucosidase activity.

(c) Strongly Basic Anion-Exchange Chromatography

In a Tris-HCl buffer (0.05 M, pH 7.0), a protein in the fractionobtained in (b) above was adsorbed to MonoQ (manufactured by GEHealthcare). The adsorbed protein was subjected to linear gradientelution in a Tris-HCl buffer (0.05 M, pH 7.0) containing 0 M to 1 M ofNaCl to fractionate a fraction indicating a β-glucosidase activity.

Example 2 Determining Partial Amino Acid Sequences of Purifiedβ-Glucosidase

The fraction having a β-glucosidase activity fractionated by thestrongly basic anion-exchange chromatography in Example 1 was separatedby electrophoresis using 12% Gel SDS-PAGE mini (manufactured by TEFCO),and β-glucosidase B (acBGLB) of Acremonium cellulolyticus wasidentified. A band of the acBGLB was cut out, and then reductivelycarboxymethylated, followed by treatment with lysyl endopeptidase. Thisdegraded product was separated by electrophoresis using 12% Gel SDS-PAGEmini (manufactured by TEFCO), and blotted on a PVDF membrane(manufactured by Millipore Corporation). A band of the peptide fragmentthus obtained was cut out. The N-terminal amino acid sequence of thepeptide fragment was determined using a protein sequencer Model 492(manufactured by Applied Biosystems Inc.). Partial amino acid sequences(“BGLB-LE-1” and “BGLB-LE-2”) of the acBGLB thus determined were shownin SEQ ID NOs: 6 and 7, respectively.

Example 3 Cloning of acBGLB Gene

(1) Isolation of Genomic DNA

An Acremonium cellulolyticus ACCP-5-1 strain was cultured in an (s)medium (2% broth, 0.5% yeast extract and 2% glucose) at 32° C. for 2days, and the fungal cells were collected by centrifugation. A genomicDNA thereof was isolated from the obtained fungal cells according to themethod by Horiuchi et al. (H. Horiuchi et. al., J. Bacteriol., 170,272-278, (1988)).

(2) Acquisition of acBGLB Gene Fragment

Based on the partial amino acid sequences of the acBGLB, the followingprimers were prepared.

BGLB-F: (SEQ ID NO: 8) CCNTTYGTNGGNAAYACNGCNGCNCC BGLB-R: (SEQ ID NO: 9)CATDATRTANCCNGGRAANCC

Using BGLB-F and BGLB-R as the primers and the genomic DNA as atemplate, PCR was performed. The PCR was performed using LA taqpolymerase (manufactured by Takara Bio Inc.). The PCR was performed in35 cycles each of “94° C. for 30 seconds, 53° C. for 30 seconds, and 72°C. for 2 minutes.” The DNA fragment of 650 bp thus amplified wasinserted into a pCR2.1-TOPO plasmid vector using TOPO TA cloning kit(manufactured by Invitrogen Corporation) in accordance with the protocolattached thereto. Thereby, a plasmid “TOPO-pBGLB-partial” was obtained.

The sequence of the inserted DNA fragment cloned in the plasmid“TOPO-pBGLB-partial” was determined using BigDye Terminator v3.1 CycleSequencing Kit (manufactured by Applied Biosystems Inc.) and ABI PRISMGenetic Analyzer (manufactured by Applied Biosystems Inc.) in accordancewith the protocols attached thereto. The base sequence thus obtained wastranslated into the amino acid sequence. Homology search was conductedon the amino acid sequence. As a result, the amino acid sequence showeda homology of 72% with the β-glucosidase (XP_001216552) derived fromAspergillus terreus and a homology of 88% with the β-glucosidase(XP_002149046.1) derived from Penicillium marneffei. Thus, the DNAfragment was determined to be a part of the β-glucosidase (GlycosideHydrolase family 3) gene.

(3) Acquisition of Full-Length acBGLB Gene by Inverse PCR Method

The inverse PCR method was performed according to the method by Trigliaet al. (T. Triglia et. al., Nucleic Acids Research, 16, 8186, (1988)).The Acremonium cellulolyticus genomic DNA was digested with ScaIovernight, and a circular DNA was prepared from the digested fragmentusing Mighty Mix (manufactured by Takara Bio Inc.). PCR was performedusing the circular DNA as a template and the following primers preparedbased on the base sequence information on the acBGLB gene fragment. A 5′upstream region and a 3′ downstream region of the acBGLB gene wereobtained.

BGLB-inv-F: (SEQ ID NO: 10) TAGGCGTTCGTTATGCGAAC BGLB-inv-R:(SEQ ID NO: 11) AAACGAGATTCCAGATGGCG

The 5′ upstream region and the 3′ downstream region were analyzed by themethod described in Example 3-(2). The full-length base sequence of theBGLB gene was determined.

Based on the base sequence obtained by the inverse PCR method, thefollowing primers were prepared. PCR was performed using the genomic DNAas a template, to amplify the BGLB gene.

pBGLB-F: (SEQ ID NO: 12) CTGGACCTATATTCCCCGAT pBGLB-R: (SEQ ID NO: 13)TGGTTTGTCCATACTGCGTC

The DNA thus amplified was inserted into a pCR2.1-TOPO plasmid vectorwith TOPO TA cloning kit (manufactured by Invitrogen Corporation) toobtain a plasmid “pBGLB.” An Escherichia coli TOP10 strain (manufacturedby Invitrogen Corporation) was transformed with the obtained plasmid“pBGLB.” Thereby, “Escherichia coli TOP10 strain/pBGLB” was obtained.

(4) Preparation of acBGLB cDNA and Intron Analysis of acBGLB Genomic DNA

An Acremonium cellulolyticus ACCP-5-1 strain was cultured in a cellulaseinduction medium at 32° C. for 2 days, and the fungal cells werecollected by centrifugation. After frozen with liquid nitrogen, theresultant fungal cells were ground using a mortor and a pestle. Thetotal RNA was isolated from the ground fungal cells with ISOGEN (NipponGene Co., Ltd.) in accordance with the protocol attached thereto.Further, a mRNA was purified from the total RNA with mRNA PurificationKit (Pharmacia Corporation) in accordance with the protocol attachedthereto.

A cDNA was synthesized from the mRNA thus obtained with TimeSaver cDNASynthesis Kit (Pharmacia Corporation) in accordance with the protocolattached thereto. The following primers containing the start codon andthe stop codon were prepared from the acBGLB gene sequence, and PCR wasperformed using the cDNA as a template.

BGLB-N: (SEQ ID NO: 14) ATGTATTCCGCATTTCTTTTGCTGC BGLB-C:(SEQ ID NO: 15) CTATTGTAGGCATTGAGAATACCAT

The base sequence (SEQ ID NO: 1) of the cDNA thus amplified was analyzedby the method described in Example 3-(2), and compared with the basesequence of the pBGLB genomic DNA. Thus, the positions of introns in thegenomic DNA were determined.

(5) Estimation of Amino Acid Sequence of acBGLB

The exons and introns of the acBGLB genomic DNA isolated by theabove-described method were composed of 2630 bp shown from positions 218to 2847 of the base sequence of SEQ ID NO: 2. Moreover, the acBGLBgenomic DNA contained three introns shown from the 734th to 792nd,1665th to 1717th, and 2523rd to 2601st of the base sequence of SEQ IDNO: 2. The amino acid sequence of the acBGLB predicted from an openreading frame (ORF) was as shown in SEQ ID NO: 3. A part of the aminoacid sequence predicted from the ORF corresponded to the internalsequence of the acBGLB determined in Example 2. This fact revealed thatthe isolated genomic DNA encoded the acBGLB. Note that, using signalsequence prediction software SignalP 3.0, the amino acid residues from−18 to −1 of the acBGLB were estimated to be a signal sequence.

Example 4 Expression of acBGLB Gene in Trichoderma viride

(1) Modification of acBGLB Gene Codon for Suitable Expression inTrichoderma viride

To express the acBGLB gene at a high level as an active protein inTrichoderma viride, the acBGLB gene was modified. As a result of trialsand errors, a DNA comprising the base sequence of SEQ ID NO: 4 which wasmodified from the acBGLB gene by 13.2% or more of the bases was foundout. In designing this modified acBGLB gene, 16 kinds of amino acidsamong 20 kinds of amino acids in the encoding base sequence weremodified; in addition, the frequency distribution of codons used inTrichoderma viride was taken into consideration. This modified acBGLBgene was artificially synthesized by Gene Design Inc. In the artificialsynthesis, the design was made such that XbaI and SnaBI were containedin a sequence upstream of the start codon, and that SalI and XbaI werecontained downstream of the stop codon. Thus, a plasmid “pBGLBkai” wasobtained in which the codon-modified acBGLB gene was inserted in XbaI ofpUC19.

(2) Construction of Codon-Modified BGLB-Expression Plasmid BGLBkai-pCB1

The plasmid “pBGLBkai” was cleaved with SnaBI and SalI, andapproximately 2.7 kbp of a gene fragment “BGLBkai-N” was obtained.Meanwhile, to remove a hygromycin B resistance cassette from pCB1-Eg3X(International Publication No. WO98/11239), the pCB1-Eg3X was cleavedwith a restriction enzyme XbaI, and then circularized again using TaKaRaDNA Ligation Kit Mighty Mix (manufactured by TAKARA SHUZO CO., LTD.).Thus, a plasmid “pCB1-Eg3X-hphless” was obtained. The“pCB1-Eg3X-hphless” was cleaved with StuI and XhoI, and approximately 7kbp of a fragment was collected. To this, approximately 2.7 kbp of thegene fragment “BGLBkai-N” was ligated using TaKaRa DNA Ligation KitMighty Mix (manufactured by TAKARA SHUZO CO., LTD.). Thus, a plasmid“BGLBkai-pCB1” was prepared. The reaction conditions such as enzymefollowed the conditions in the protocol attached to the kit. The plasmid“BGLBkai-pCB1” was constructed in such a manner as to express themodified acBGLB using the start codon of its own in the host Trichodermaviride.

(3) Preparation of Trichoderma viride Transformant with Plasmid“BGLBkai-pCB1”

Trichoderma viride was transformed with the plasmid “BGLBkai-pCB1”obtained in Example 4-(2) in accordance with the method described inInternational Publication No. WO2005/056787. The transformation wascarried out by a co-transformation method using Trichoderma viridestrain 2 deficient in a gene for uracil biosynthesis (pyr4) as a hostand a pyr4 gene of Neurospora crassa as a selection marker. TheTrichoderma viride strain 2 was cultured in 50 mL of a fungalcell-forming medium (1% yeast extract, 1% malt extract, 2% polypeptone,2.5% glucose, 0.1% dipotassium hydrogen phosphate, 0.05% magnesiumsulfate heptahydrate, 0.0001% uridine (pH7.0)) at 28° C. for 24 hours,and centrifuged at 3000 rpm for 10 minutes, and the fungal cells werecollected. The obtained fungal cells were washed with 0.5 mol/L ofsucrose, and suspended in a protoplast-forming enzyme solution (1 mg/mLof β-glucuronidase, 0.3 mg/mL of chitinase, 0.3 mg/mL of zymolyase, 0.5mol/L of sucrose) that had been filtered through cotton. The mixture wasshaken at 30° C. for 60 minutes, so that the hypha was formed into aprotoplast. This suspension was filtered, and then centrifuged at 2500rpm for 10 minutes to collect the protoplast, which was washed with aSUTC buffer (0.5 mol/L of sucrose, 10 mmol/L of calcium chloride, 10mmol/L of Tris-HCl (pH 7.5)).

The protoplast was suspended in 100 μL of a SUTC buffer, to which 10 μgof a DNA solution 10 μL containing the plasmid “BGLBkai-pCB1” and 10 μLof a DNA solution containing the pyr4 gene were added. The resultant wasallowed to stand in ice for 5 minutes. Next, 400 μl of a PEG solution(60% of PEG4000, 10 mmol/L of calcium chloride, 10 mmol/L of Tris-HCl(pH 7.5)) was added thereto, which was allowed to stand in ice for 20minutes. Then, 10 mL of a SUTC buffer was added thereto, which wascentrifuged at 2500 rpm for 10 minutes. The protoplast thus collectedwas suspended in 1 mL of a SUTC buffer, and each 200-μl solution of theprotoplast suspension was overlaid with soft agar on a minimum mediumcontaining 0.5 mol/L of sucrose, followed by culturing at 28° C. for 5days. Subsequently, grown colonies were again transferred on a minimummedium. The colonies formed thereon were used as transformants.

(4) Culturing and Identification of “BGLBkai-pCB1” Transformant

The plasmid “BGLBkai-pCB1” was introduced into each of the minimummedia. A line grown on the medium was selected, and cultured inaccordance with WO 98/11239 A. The resultant culture supernatant fluidwas separated by electrophoresis using 12% Gel SDS-PAGE mini(manufactured by TEFCO). A culture supernatant having a favorablydetectable band which migrated the same distance as that of the acBGLBidentified in Example 2 was selected.

(5) Identification of Partial Amino Acid Sequence of RecombinantModified acBGLB

To confirm that the protein expressed in a large amount in Example 4-(4)was the modified acBGLB, the partial amino acid sequence was determined.First, the protein in the culture supernatant was separated byelectrophoresis using 12% Gel SDS-PAGE mini (manufactured by TEFCO). Aband corresponding to the acBGLB separated according to the method inExample 2 was treated with lysyl endopeptidase. The degraded product wasseparated by electrophoresis using 12% Gel SDS-PAGE mini (manufacturedby TEFCO), and blotted on a PVDF membrane (manufactured by MilliporeCorporation). A band of the peptide fragment thus obtained was cut out.The N-terminal amino acid sequence of the peptide fragment wasdetermined using a protein sequencer Model 492 (manufactured by AppliedBiosystems Inc.). As a result, the N-terminal amino acid sequencecorresponded to the partial amino acid sequence (SEQ ID NO: 6) of theacBGLB.

Example 5 Measurement of Enzyme Activity of Trichoderma virideTransformant

The β-glucosidase activity was measured using the culture supernatant ofthe “BGLBkai-pCB1” transformant obtained in Example 4-(4). Themeasurement method followed the method described in the literature(Methods in ENZYMOLOGY, vol. 160, Biomass Part A Cellulose andHemicellulose, Willis A. Wood ed. p 109-110). Note that theβ-glucosidase activity is defined as an activity of producing 1 μmol ofp-nitrophenol from p-nitrophenyl-β-glucoside in 1 minute, and wasrepresented as activity (U/mL) per mL of the culture supernatant. Theresult is as shown in Table 1. As apparent from Table 1, thetransformant showed the activity approximately 7.5 times as high as aparental strain (original Trichoderma viride strain not deficient in agene for uracil biosynthesis).

TABLE 1 β-glucosidase activity (U/mL) Parental strain  211 Transformant1592

This revealed that a cellulase-producing microorganism having a lowβ-glucosidase activity over-expressed a β-glucosidase derived fromAcremonium cellulolyticus, and that it was thus made possible to enhancethe β-glucosidase activity of the microorganism.

INDUSTRIAL APPLICABILITY

As described above, the present invention makes it possible to obtain aβ-glucosidase derived from Acremonium cellulolyticus as a purifiedprotein or a cellulase preparation in high yield. By using theβ-glucosidase or the cellulase preparation thus obtained,saccharification from biomass to glucose can be promoted, and treatmentsand modifications on a cellulose-based substrate can be carried outefficiently. Moreover, these β-glucosidase and cellulase preparation canbe utilized at reduced cost. Further, by utilizing a filamentous fungusexpressing a reduced amount of the β-glucosidase of the presentinvention, cellobiose useful as a sweetener, a cosmetic raw material, ora drug raw material can be produced efficiently.

The invention claimed is:
 1. A protein comprising: any one of thefollowing (i) to (ii): (i) the amino acid sequence of SEQ ID NO: 3 butin which 1 to 8 amino acids are substituted, deleted, inserted and/oradded; and (ii) the amino acid sequence of (i) from which a signalsequence is removed, provided that said protein has β-glucosidaseactivity and contains at least one amino acid insertion or substitutionrelative to the sequence of residues 1-795 of SEQ ID NO: 3, wherein saidprotein has been produced through being expressed from a filamentousfungus belonging to the genus Trichoderma.
 2. The protein according toclaim 1, which is a recombinant protein.
 3. A cellulase preparationcomprising a protein comprising any one of the following (i) to (ii):(i) the amino acid sequence of SEQ ID NO: 3 but in which 1 to 8 aminoacids are substituted, deleted, inserted and/or added; and (ii) theamino acid sequence of (i) from which a signal sequence is removed,provided that said protein has β-glucosidase activity and contains atleast one amino acid insertion or substitution relative to the sequenceof residues 1-795 of SEQ ID NO: 3, wherein said protein has beenproduced through being expressed from a filamentous fungus belonging tothe genus Trichoderma, and another protein, wherein the another proteinis at least one protein selected from the group consisting ofhemicellulase, endoglucanase, cellobiohydrolase, aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinase, peptide glutaminase, peroxidase, phytase, polyphenol oxidase,protease, ribonuclease, transglutaminase, and xylanase.
 4. A detergentcomposition comprising a surfactant in admixture with a substanceselected from any one of the following (a) to (b): (a) the proteinaccording to claim 1, and (b) a cellulase preparation comprising aprotein comprising any one of the following (i) to (ii) in admixturewith another protein: (i) the amino acid sequence of SEQ ID NO: 3 but inwhich 1 to 8 amino acids are substituted, deleted, inserted and/oradded; and (ii) the amino acid sequence of (i) from which a signalsequence is removed, provided that said protein has β-glucosidaseactivity and contains at least one amino acid insertion or substitutionrelative to the sequence of residues 1-795 of SEQ ID NO: 3, wherein saidprotein has been produced through being expressed from a filamentousfungus belonging to the genus Trichoderma, wherein the another proteinis at least one protein selected from the group consisting ofhemicellulase, endoglucanase, cellobiohydrolase, aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinase, peptide glutaminase, peroxidase, phytase, polyphenol oxidase,protease, ribonuclease, transglutaminase, and xylanase.
 5. A method fordegrading or converting a cellulose material, the method comprising thestep of: treating the cellulose material with a substance selected fromany one of the following (a) to (b): (a) the protein according to claim1, and (b) a cellulase preparation comprising a protein comprising anyone of the following (i) to (ii) in admixture with another protein: (i)the amino acid sequence of SEQ ID NO: 3 but in which 1 to 8 amino acidsare substituted, deleted, inserted and/or added; and (ii) the amino acidsequence of any (i) from which a signal sequence is removed, providedthat said protein has β-glucosidase activity and contains at least oneamino acid insertion or substitution relative to the sequence ofresidues 1-795 of SEQ ID NO: 3, wherein said protein has been producedthrough being expressed from a filamentous fungus belonging to the genusTrichoderma, wherein the another protein is at least one proteinselected from the group consisting of hemicellulase, endoglucanase,cellobiohydrolase, aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, α-galactosidase,β-galactosidase, glucoamylase, α-glucosidase, haloperoxidase, invertase,laccase, lipase, mannosidase, oxidase, pectinase, peptide glutaminase,peroxidase, phytase, polyphenol oxidase, protease, ribonuclease,transglutaminase, and xylanase.
 6. A method for producing a degraded orconverted cellulose material, the method comprising the steps of:treating a cellulose material with a substance selected from any one ofthe following (a) to (b): (a) the protein according to claim 1, and (b)a cellulase preparation comprising a protein comprising any one of thefollowing (i) to (ii) in admixture with another protein: (i) the aminoacid sequence of SEQ ID NO: 3 but in which 1 to 8 amino acids aresubstituted, deleted, inserted and/or added; and (ii) the amino acidsequence of (i) from which a signal sequence is removed, provided thatsaid protein has β-glucosidase activity and contains at least one aminoacid insertion or substitution relative to the sequence of residues1-795 of SEQ ID NO: 3, wherein said protein has been produced throughbeing expressed from a filamentous fungus belonging to the genusTrichoderma, wherein the another protein is at least one proteinselected from the group consisting of hemicellulase, endoglucanase,cellobiohydrolase, aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, α-galactosidase,β-galactosidase, glucoamylase, α-glucosidase, haloperoxidase, invertase,laccase, lipase, mannosidase, oxidase, pectinase, peptide glutaminase,peroxidase, phytase, polyphenol oxidase, protease, ribonuclease,transglutaminase, and xylanase; and collecting a degraded cellulosematerial.
 7. The method according to claim 6, wherein the degradedcellulose material is a sugar.
 8. A method for treating acellulose-containing fiber, the method comprising the step of: bringinga substance selected from any one of the following (a) to (d) intocontact with the cellulose-containing fiber: (a) the protein accordingto claim 1, and (b) a cellulase preparation comprising a proteincomprising any one of the following (i) to (ii) in admixture withanother protein: (i) the amino acid sequence of SEQ ID NO: 3 but inwhich 1 to 8 amino acids are substituted, deleted, inserted and/oradded; and (ii) the amino acid sequence of any (i) from which a signalsequence is removed, provided that said protein has β-glucosidaseactivity and contains at least one amino acid insertion or substitutionrelative to the sequence of residues 1-795 of SEQ ID NO: 3, wherein saidprotein has been produced through being expressed from a filamentousfungus belonging to the genus Trichoderma, (c) a detergent compositioncomprising a protein comprising any one of the following (i) to (ii) inadmixture with a surfactant: (i) the amino acid sequence of SEQ ID NO: 3but in which 1 to 8 amino acids are substituted, deleted, insertedand/or added; and (ii) the amino acid sequence of (i) from which asignal sequence is removed, provided that said protein has β-glucosidaseactivity and contains at least one amino acid insertion or substitutionrelative to the sequence of residues 1-795 of SEQ ID NO: 3, wherein saidprotein has been produced through being expressed from a filamentousfungus belonging to the genus Trichoderma, and (d) a detergentcomposition comprising a protein comprising any one of the following (i)to (ii) in admixture with another protein and a surfactant: (i) theamino acid sequence of SEQ ID NO: 3 but in which 1 to 8 amino acids aresubstituted, deleted, inserted and/or added; and (ii) the amino acidsequence of (i) from which a signal sequence is removed, provided thatsaid protein has β-glucosidase activity and contains at least one aminoacid insertion or substitution relative to the sequence of residues1-795 of SEQ ID NO: 3, wherein said protein has been produced throughbeing expressed from a filamentous fungus belonging to the genusTrichoderma, and wherein the another protein is at least one proteinselected from the group consisting of hemicellulase, endoglucanase,cellobiohydrolase, aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, α-galactosidase,β-galactosidase, glucoamylase, α-glucosidase, haloperoxidase, invertase,laccase, lipase, mannosidase, oxidase, pectinase, peptide glutaminase,peroxidase, phytase, polyphenol oxidase, protease, ribonuclease,transglutaminase, and xylanase.
 9. A method for producing an animal feedhaving an improved digestibility, the method comprising the step of:treating an animal feed with a substance selected from any one of thefollowing (a) to (b): (a) the protein according to claim 1, and (b) acellulase preparation comprising a protein comprising any one of thefollowing (i) to (ii) in admixture with another protein: (i) the aminoacid sequence of SEQ ID NO: 3 but in which 1 to 8 amino acids aresubstituted, deleted, inserted and/or added; and (ii) the amino acidsequence of (i) from which a signal sequence is removed, provided thatsaid protein has β-glucosidase activity and contains at least one aminoacid insertion or substitution relative to the sequence of residues1-795 of SEQ ID NO: 3, wherein said protein has been produced throughbeing expressed from a filamentous fungus belonging to the genusTrichoderma, wherein the another protein is at least one proteinselected from the group consisting of hemicellulase, endoglucanase,cellobiohydrolase, aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, α-galactosidase,β-galactosidase, glucoamylase, α-glucosidase, haloperoxidase, invertase,laccase, lipase, mannosidase, oxidase, pectinase, peptide glutaminase,peroxidase, phytase, polyphenol oxidase, protease, ribonuclease,transglutaminase, and xylanase.